Publikationsrepositorium - Helmholtz-Zentrum Dresden-Rossendorf

Introduction and Aim:

Phosphopeptides are very useful reagents to study signal transduction pathways related with cellular protein phosphorylation/dephosphorylation processes. Phosphopeptides also have been identified as important drug candidates to modulate intracellular signal transduction pathways. The present study describes the use of cell-penetrating peptides (CPPs) sC18 and hCT(18-32)-k7 coupled to Polo-like kinase-1 (Plk-1)-binding hexaphosphopeptide H-Met-Gln-Ser-pThr-Pro-Leu-OH to investigate cell uptake of the corresponding phosphopeptide-CPP hybrids.

Materials and Methods:

Peptide syntheses were accomplished using a combination of automated and manual solid-phase peptide syntheses following standard Fmoc chemistry. Fluorescence labeling was carried out via coupling 5(6)-carboxyfluoresceine (CF) to the N-terminal end of the phosphopeptide using HATU as the activation agent, DIPEA as the base, and DMF as the solvent. Cell uptake studies of CF-Met-Gln-Ser-pThr-Pro-Leu-sC18 (CF-I) and CF-Met-Gln-Ser-pThr-Pro-Leu-hCT(18-32)-k7 (CF-II) were performed using the human breast cancer cell line MCF-7 and human cervical carcinoma epithelial (HeLa) cells on a Zeiss Axio fluorescence confocal microscope. Results: Peptide hybrids CF-I and CF-II showed cellular uptake in both cell lines after incubation at 37°C for 60 min. Cell uptake was more pronounced in HeLa cells compared to MCF-7 cells. Phosphopeptide conjugated to sC18 showed higher cell uptake compared to phosphopeptide-hCT(18-32)-k7 conjugate. As expected, CF-labeled phosphopeptide without being conjugated to CPPs as molecular shuttles was not internalized into both cell lines. Quantification of cellular uptake using flow cytometry analysis and fluorine-18 labeled phosphopeptide-CPP hybrids are currently in progress.

Conclusion:

Cell-penetrating peptides sC18 and hCT(18-32)-k7 are useful drug delivery systems enabling sufficient membrane transport of phosphopeptides to further promote studies on intracellular metabolic pathways involving phosphopeptides.